Bacillus cereus strain MS1-9, ATCC 55812

ABSTRACT

A novel strain of Bacillus cereus, designated MS1-9, has been isolated from the environment. The strain MS1-9, ATCC 55812, is one of a number of B. cereus strains which are useful as biocontrol agents to combat fungal damping off disease in field crop plants, and strain MS1-9 has exhibited good performance under high disease pressure among a large number of natural isolates in fostering the emergence and growth of alfalfa plants under normal field conditions in the upper midwestern U.S.

BACKGROUND OF THE INVENTION

The present invention is in the general field of bacteriology and relates, in particular, to a novel strain of bacteria useful as a biocontrol agent in field applications.

Significant research has been conducted in recent years on the use of biological agents to increase agricultural productivity and efficiency. Biological control based on the use of microorganisms to suppress plant pests or supplement plant growth offers an attractive alternative to chemical pesticides which are less favored than they have previously been because of concerns about human health and environmental quality. Several screening programs have been used before to isolate biological agents which are effective in the laboratory or in the field to combat pests or facilitate plant growth.

An example of a biological control agent into which significant scientific and economic development has occurred is the use of the Bacillus thuringiensis. It was recognized that B. thuringiensis strains produced toxic proteins which have the ability to specifically kill certain insects and that initial inquiry led to a significant research which has proceeded to identify a large number of B. thuringiensis strains having variations and target range in efficacy. In addition, research has been conducted on methods for stabilizing and applying such toxins, or strains harboring them, to a wide variety of field crop situations. It was also discovered that knowledge of B. thuringiensis strains was largely transferable to new strains since the toxins required for biological control and methods for preparing inocula for use in the field were generally similar among strains.

Previously it has been found that a specific strain of Bacillus cereus, which has been referred to both as UW85 and by its ATCC designation 53522 has biocontrol efficacy in many applications. The UW85 B. cereus strain was found to protect alfalfa seedlings from damping off caused by Phytopthora medicaginis, tobacco seedlings from Phytopthora nicotianae, cucumber fruits from rot caused by Pythium aphanidermatum, and peanuts from Sclerotinia minor. UW85 is also described, by reference to its ATCC number in U.S. Pat. No. 4,877,738. It was later found that UW85 produced two antifungal compounds which contribute independently to its suppression of damping off fungi due to antifungal and antibacterial activity. The more potent of these compounds, a novel aminopolyol has been designated zwittermicin A while the second compound, not well characterized, was provisionally designated antibiotic B and is now known to be kanosamine.

"Biological control" is defined as pathogen suppression by the use of a second organism. Mechanisms of biological Control are diverse. For example, certain enteric bacteria have been examined for their usefulness in biological control of root rot in alfalfa. It is believed that control is obtained by competition between the enteric bacteria and the fungi for space on the surface of the alfalfa roots. In contrast, a toxin produced by one species of bacteria may be used to control another species of bacteria that appears as a pathogen. Bacterially produced antibiotics are an example of such toxins. The toxin can be isolated from the species producing it and administered directly, as is the common procedure with penicillin, or the species itself may be administered under appropriate circumstances to produce the toxin in situ. Once identified, such toxins produced by soil-dwelling bacteria may have utility in diverse other areas as antifungal or antibiotic agents.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that a novel Bacillus cereus strain, here designated MS1-9, ATCC No. 55812, has been isolated from the environment. Strain MS1-9 has been found to have increased efficacy in fostering the growth and establishment of alfalfa plants in field environments of the upper mid-western United States where root rot diseases of alfalfa are severe.

The present invention is further characterized in that a method is described to foster the growth of alfalfa stands by the application of an inoculum including as its active agents a novel Bacillus cereus isolate designated MS1-9, ATCC No. 55812.

Other objects, advantages, and features of the present invention will become apparent from the following specification.

DETAILED DESCRIPTION OF THE INVENTION

An original bacterial strain, isolated from soil, exerts biological control over species of fungi responsible for damping off and root rot in plants. This strain is particularly adapted for use in soils having severe alfalfa root rot problems. The strain has been deposited in the American Type Culture Collection, Rockville, Md., on Aug. 30, 1996 and given the designation ATCC 55812, and shall hereinafter be referred to either as Strain MS1-9 or ATCC 55812.

It is further anticipated that certain mutants of MS1-9 also provide biological control comparable to that provided by MS1-9 in severe root rot conditions. These bacterial strains may also be obtained in substantially pure cultures. A "substantially pure" culture shall be deemed a culture of a bacteria, containing no other bacterial species in quantities sufficient to interfere with replication of the culture or to be detectable by normal bacteriological techniques. In addition, it has been discovered that the biological control is exerted principally by means of one or more toxins produced by the bacterial strain.

Strain MS1-9 is one of a group of Bacillus cereus strains that are useful biocontrol agents due, at least in part, to the fact that they naturally synthesize antibiotic agents, notably an antibiotic which is the subject of a co-pending patent application. The antibiotic or toxin is found in supernatant fluid and other bacteria-free fluid and culture medium removed from a culture of MS1-9 or of its protecting mutants, has been found to be a "protecting toxin," as that term is defined below. This toxin has been so characterized as to be identifiable independent of its source in cultures of Bacillus cereus, and is known and by the coined term "zwittermicin A." Another fraction from the supernatant fluid from a culture of B. cereus ATCC 53522 has been found biologically active, having a zoospore lysis capability to zoospores of Phytopthera medicaginis (Pmm), but, as revealed below, this zoospore lysis active fraction does not have the antifungal activity of the antibiotic. Bacillus cereus antibiotic zwittermicin A has been found to be a highly water soluble molecule of about 396 daltons. The molecule has two amino groups, and is a linear poly-alcohol.

The method by which the biological control referred to in the preceding paragraph may be verified to exist is the "plant protection assay" detailed below. "Biological control" of fungi causing damping off and root rot shall be deemed to exist if, when an effective quantity of MS1-9, its mutants that exhibit biological control, the anti-fungal toxin produced by them, or any other compound or molecule is placed in the soil or other growing medium in the immediate vicinity of the plant to be protected, a statistically significant reduction in the symptoms of damping off or root rot occurs in the presence of one or more pathogens causing these diseases. An "effective quantity" to combat damping off and root rot shall be that quantity sufficient to result in such a visibly significant reduction of symptoms. Clearly, if no quantity of a bacteria or any toxin or other compound is an effective quantity as so defined, that bacteria, toxin, or compound is not capable of exerting biological control over the fungi causing damping off and root rot.

MS1-9 and those of its mutants capable of exerting such biological control shall sometimes be referred to collectively as "protecting" bacteria. Bacillus cereus antibiotic and other toxins capable of exerting such biological control shall sometimes be referred to as "protecting" compounds or toxins. Plants, including seeds, seedlings, and mature plants, treated with such an effective quantity of protecting bacteria, their toxins, or Bacillus cereus antibiotic shall be referred to as "protected" from root rot or damping off.

The following is a disclosure of the plant protection assay whereby a test material such as a bacteria, a toxin, or the like, may be tested for its ability to exert biological control over a fungus capable of causing the symptoms of damping off or root rot. The seed or seedling of the plant to be protected is planted in a planting medium in the presence of damping off or root rot causing fungi. The planting medium may be a damp soil containing such fungi, vermiculite in water with the fungi present either in the vermiculite and water or in or on the seed or seedling, an agar-based formulation, or any other planting medium in which the seed or seedling will grow and the fungi may freely develop. The bacteria, toxin, or other test material is placed at least in the immediate vicinity of the seed or seedling. Such placement shall be understood to be in the "immediate vicinity" of the seed or seedling if any soluble test material or any soluble exudate of a bacteria being tested will be in actual contact with the germinating seedling.

Preferably, if seed is used, the seed is coated with the test material, and when the test material is so used with respect to a seed, it shall be referred to hereinafter as a "seed inoculum." The process of coating seed with a seed inoculum is generally well known to those skilled in the art, and any conventional method that does not require conditions sufficiently harsh to kill bacteria, to harm the seeds, or to destroy toxins or other materials included in the seed inoculum, is adequate. An easy and preferred method is to apply the bacteria directly to the seed and dry the seed under vacuum.

After a time sufficient for seedling growth and the expression of the symptoms of damping off, seedlings developing from the planted seed may be evaluated for visual evidence of protection, when compared to controls. In strains of alfalfa, soybeans, and snap beans known to be vulnerable to damping off, one to two weeks of growing time in a growth chamber at 24° C. with a 12 hour photoperiod was found to be a period sufficient for the expression of symptoms of damping off when seedlings were being grown in test tubes containing roughly 10³ zoospores of Pmm or comparable, damping off-causing fungi. Protected seeds developed into seedlings visually indistinguishable from uninfected seeds while control seedlings developing from unprotected seeds were killed or, in the case of snap beans, exhibited brown lesions on roots and stems, stunted roots, rotted roots, and other visually apparent symptoms of root rot.

Protecting mutants of MS1-9 include both naturally occurring and artificially induced mutants. For example, MS1-9 is generally sensitive to the antibiotics rifampicin and neomycin. However, it is expected that naturally occurring mutants of MS1-9 can be isolated that exhibited resistance to one or the other of these antibiotics. Certain of these mutants, as well as one naturally occurring mutant distinguishable from the parent MS1-9 strain by the appearance of its colonies, will be found to protect alfalfa plants in the plant protection assay. Other mutants of MS1-9 can be artificially induced by subjecting MS1-9 to the mutagen N-methyl-nitrogoguanidine in conventional ways. Similar mutants have been made from other useful B. cereus strains, such as UW85 (ATCC 53522), as described in U.S. Pat. No. 4,877,738, the disclosure of which is hereby incorporated by reference.

In terms of conventional bacteriological morphology and colony analysis techniques, strain MS1-9, ATCC 55812, is indistinguishable from UW85, ATCC 53522 or from other useful B. cereus biocontrol strains such as AS4-12, ATCC 55609. Strain MS1-9 is sensitive in the P7 phage assay, described below, scores very high on the Erwinia inhibition laboratory assay, also described below, scores high on bioassay, and is positive for the production of both zwittermicin A and the antibiotic formerly referred to as antibiotic B, now known to be kanosamine. The differences between the strains largely relate to the statistical difference in actual field performance conditions, as reported below. The strain MS1-9 has proven to be particularly adapted to those field situations in which alfalfa root rot is especially severe. While on average, over all fields, the performance of strain AS4-12 is better, strain MS1-9 was the only other strain to provide significant and repeatable yield enhancement in fields, such as those in the UW experimental plot at Marshfield, Wisconsin, where alfalfa root rot infestation was severe.

EXAMPLE 1 Origin of Strains

The geographic origins, and physical and chemical characteristics of the soil samples used in this study are listed in Table 1. All samples were taken from the surface horizon. Measurements of soil pH, organic matter, and particle size were made by the University of Wisconsin Soil & Plant Analysis Laboratory (Madison, Wis.).

                  TABLE 1                                                          ______________________________________                                         Characteristics of soils used in this study                                                                            Most                                                                    % Sand,                                                                               recent                                                                  Silt,  vege-                                  Soil/Site                                                                               Country   pH    % Organic.sup.a                                                                        Clay.sup.b                                                                            tation                                 ______________________________________                                         Lutz     Panama    nd.sup.c                                                                             5.5     nd     Forest                                 Snyder-Molino                                                                           Panama    nd    6.2     nd     Forest                                 Barbour- Panama    nd    7.1     nd     Forest                                 Lathrop                                                                        Moroceli Honduras  nd    1.7     nd     Maize-                                                                         Beans                                  San Matias                                                                              honduras  5.8   1.5     nd     Maize-                                                                         Beans                                  LaVega1  Honduras  6.3   2.3     nd     Beans                                  LaVega5  Honduras  5.9   2.5     nd     Sorghum-                                                                       Maize                                  Arlington WI                                                                            USA       6.9   4.2     23, 68, 9                                                                             Alfalfa                                Hancock WI                                                                              USA       6.6   0.8     87, 8, 5                                                                              Alfalfa-                                                                       Oats                                   Marshfield WI                                                                           USA       6.5   3.1     23, 72, 5                                                                             Alfalfa                                Lancaster WI                                                                            USA       7.1   2.3     19, 68, 13                                                                            Alfalfa                                Madison WI                                                                              USA       7.0   3.2     27, 56, 17                                                                            Alfalfa                                Taos NM  USA       8.1   4.3     nd     Pasture                                Tifton GA                                                                               USA       6.3   1.0     87, 12, 1                                                                             Tobacco                                Douglas Gully                                                                           Austrailia                                                                               6.1   2.2     nd     Vineyard                               Lelystad Netherlands                                                                              7.5   1.8     51, 36, 13                                                                            Potatoes                               ______________________________________                                    

Isolation and Identification of B. cereus Isolates

The bacterial strains and isolates and their origins are listed in Table 2. Bacteria collected from soybean roots were isolated as previously described from plants grown in a field plot in Madison, Wis. (Table 2). The remaining bacteria collected in this study were isolated by placing either an entire alfalfa root or 1 g of soil in a test tube containing 9 ml water and sonicating it for 30 sec. in a Model 2200 bath sonicator (Branson Ultrasonics Corp., Danbury, Conn.), Serial 10-fold dilutions of the suspensions were made in water, and 0.1 ml from dilutions ranging from 10⁻¹ to 10⁻⁵ were spread on either 1/10-strength trypticase soy agar (1/10-strength TSA) (Becton Dickinson Microbiology Systems, Cockeysville Md.) or MinIC media. Plates were incubated at room temperature or 28° C. for one to three days. The plates containing isolated colonies were used for further study. Colonies that had the morphology typical of B. cereus (flat, broad, and cream colored) were picked and streaked for isolated colonies. As a partial selection for B. cereus, during either the initial plating or subsequent streak plating, the medium was supplemented with polymyxin (25 μg/ml), cycloheximide (100 μg/ml), and ampicillin (50 μg/ml). All isolates were tested for hemolysis of blood agar, which is diagnostic of B. cereus, and those that were non-hemolytic were removed from the collection. Blood agar was obtained from the Wisconsin State Hygiene Lab, Madison, Wis. Isolates were stored on 1/2-strength trypticase soy agar (1/2-strength TSA) slants. Alfalfa plants were grown from seed in soil from Arlington Wis. for 21 days in a growth chamber at 24° C. with a 12 hour photoperiod and a light intensity of 244 microeinsteins/m² /s. Soybean plants were grown from seed in a field plot in Madison Wis.

                                      TABLE 2                                      __________________________________________________________________________     Strains and isolates used in this study                                        Strain(s)/Isolates    Origin                                                   __________________________________________________________________________     ATCC7064, ATCC27877, ATCC12826                                                                       American Type Culture Collection                         BGSC6A3, BGSC6E1, BGSC6E2, BGSC4A9,                                                                  Bacillus Genetic Stock Center                            BGSC4B1, BGSC4C3, HD1, BGSC4E1, BGSC4F1,                                       BGSC4G1, BGSC4H1, BGSC4I1, BGSC4J1,                                            BGSC4S2                                                                        T                     U. W. Bacteriology Dept. Collection                      UW85                  Alfalfa root, Arlington, WI (26)                         Soy130                Soybean root, Walnut St. Farm,                                                 Madison, WI                                              ALF1, ALF9, ALF10, ALF13, ALF19, ALF23,                                                              Roots of alfalfa plants planted                          ALF52, ALF53, ALF79, ALF83, ALF85, ALF94,                                                            in soil from Arlington, WI and                           ALF95, ALF98, ALF99, ALF108, ALF109,                                                                 grown in growth chamber                                  AFL115, ALF117, ALF133, ALF137, ALF144,                                        ALF154, ALF157, ALF161, ALF166, ALF167,                                        ALF173                                                                         LUTZ21, LUTZ58, LUTZ128                                                                              Lutz soil                                                SNY14, SNY42, SNY44, SNY45, SNY73                                                                    Snyder-Molino soil                                       BAR78, BAR145, BAR177 Barbour-Lathrop soil                                     MOR1, MOR28, MOR37    Moroceli soil                                            SM32, SM43, SM44      San Matias soil                                          VGA19, VGA118, VGA137 LaVega1 soil                                             VGA562, VGA577, VGA598                                                                               LaVega5 soil                                             AS7-4, AS8-4, AG8-13, AS4-12, ARL8                                                                   Arlington soil                                           HS1-3, HS23-11, HS24-8, HS2409                                                                       Hancock soil                                             MS1-9, MS3-2, MS8-2   Marshfield soil                                          LS2-2, LS2-12, LS33-2 Lancaster soil                                           WS4-12, WS8-8, WS10-15, WS16-4, WS22-12                                                              Madison soil                                             TNM68, TNM155, TNM243 Taos soil                                                TG38, TG42, TG126     Tifton soil                                              DGA34, DGA37, DGA84, DGA94                                                                           Douglas Gully soil                                       LN24, LN75, LN100     Lelystad soil                                            __________________________________________________________________________

Based on the profiles of fatty acids from 47 isolates analyzed by five Star Labs (Branford Conn.) and Microbial ID (Newark Del.), all of the isolates were classified as members of the B. cereus group, which includes the species B. mycoides, B. anthracis and B. thuringiensis. The unique rhizoidal morphology of B. mycoides strains differentiates them from B. cereus, and none of the isolates in this collection display B. mycoides-like morphology. B. anthracis is not hemolytic and is usually sensitive to ampicillin and therefore was probably excluded from this collection. Differentiation between B. cereus and B. thuringiensis is difficult with standard methods. Therefore we have followed current recommendations and considered all isolates gathered in this study as B. cereus. Strains BGSC4A9, BGSC4B1, BGSC4C3, HD1, BGSC4E1, BGSC4F1, BGSC4G1, BGSC4H1, BTSC4I1, BGSC4J1 and BGSC4S2 were previously classified by others as B. thuringiensis, and that species designation was retained for those strains.

Assay for Sensitivity to Phage P7

The phages P7 (ATCC 75237) and PB were used to help characterize the strains. The susceptibility of B. cereus strains to infection by phage P7 has proven to have a strong co-relation to biocontrol utility and antibiotic production. To propagate these phages, we spread a mixture of melted soft agar (4 g agar/l) with approximately 10⁶ PFU of phage and an excess of B. cereus strain UW85 on 1/2-strength TSA plates. Plates were incubated overnight at 28° C. and then the soft agar was scraped off the plates and suspended in 1/2-strength trypticase soy broth (1/2-strength TSB), (1 ml/plate). Agar and cells were removed by centrifugation, and the supernatant solution was passed through a 0.2 μm filter. Phage titers were typically 1×10¹⁰ PFU/ml.

To screen large numbers of isolates for P7 sensitivity, grids of 48 isolates were grown on 1/10-strength TSA and then cells were transferred with a metal replicator onto 1/10-strength TSA plates that had been spread with dilutions of P7 such that they contained approximately 10⁸, 10⁴, and 10³ PFU/plate. A 1/10-strength TSA plate containing no phage was used as a control. Isolates that appeared to form patches with decreased growth or plaques on plates containing P7 were tested in the soft-agar overlay assay (described below) to determine if they were P7^(s). Most isolates that were P7^(r) in the primary screen were not re-tested.

In the second test for sensitivity of bacterial isolates to P7, each isolate was grown on 1/2-strength TSA and cells were scraped off plates and mixed in soft agar overlays to form lawns on fresh 1/2-strength TSA plates. Ten-fold dilutions of P7 were placed in 5-μl drops on the plates, which were then incubated at 28° C. If plaques appeared, the strain was designated P7-sensitive (P7^(s)). Lawns of two isolates, ARL8 and HS23-11, were cleared by undiluted drops of P7, but P7 did not form isolated plaques on these isolates at lower concentrations. The clearing due to high titer drops appeared to be due to P7 rather than a chemical present in UW85 lysates, since high titer drops of lysates of PB, which produces turbid plaques on UW85, did not cause clearing on lawns of ARL8 and HS23-11. Therefore these strains were also scored P7^(s). Isolates whose lawns appeared unaffected by P7 were scored P7^(r).

Assay for Inhibition of Erwinia herbicola

Inhibition of E. herbicola LS005 was assayed as described in Silo-Sub et al. Appl. Environ. Microbiol., 60:2023-2030 (1994), with the following modifications. Three-day-old cultures of each B. cereus isolate grown in 1/2-strength TSB were tested to determine whether they inhibited E. herbicola on 1/1000-strength TSA plates. Isolates that produced visible zones of inhibition of E. herbicola were tested again. Isolates that produced visible zones of inhibition in both tests were scored Eh⁺. Isolates that did not noticeably inhibit E. herbicola in each of two initial tests were scored Eh⁻. Some B. cereus isolates did not inhibit E. herbicola during initial testing but did after storage at -20° C., and certain isolates (ALF115,HD1 and BGSC4S2) had variable phenotypes producing either small zones of inhibition or no zone in subsequent tests; these were classified Eh⁻.

Assay for Zwittermicin A and Antibiotic B

Zwittermicin A and antibiotic B (kanosamine) were identified in culture supernatants by cation exchange chromatography using CM SEP-PAK cartridges (Millipore, Millford, Mass.) followed by high voltage paper electrophoresis (MVPE). The cation fraction from the equivalent of 4 ml of culture supernatant was applied to the paper, which was stained with silver nitrate after electrophoresis, described in Silo-Suh et al. supra. Isolates that produced material indistinguishable from either authentic zwittermicin A or authentic kanosamine in HVPE were designated zwittermicin A producers or kanosamine producers, respectively. To verify the structural identity of zwittermicin A produced by nine representatives of the collection of isolates, putative-zwittermicin A was purified from these isolates, and subjected to proton nuclear magnetic resonance spectroscopy (¹ H-NMR) and fast atom bombardment mass spectrometry.

Assay for Suppression of Alfalfa Damping-off

Bacterial isolates were grown for three days in 1/2-strength TSB and tested in an assay for damping-off. Each isolate was tested on fifteen plants in each of 7 separate experiments with the following exceptions: strain ATCC12826 was omitted from experiments 1 and 2; strain BAR78 was omitted from experiment 4; and strains WS8-8 and LS2-12 were omitted from experiment 5. Statistical analyses (analysis of variance, Dunnet's comparison test, standard error of least squared mean) were conducted using the SAS Computer Program (SAS Institute, Raleigh, N.C.). The data from the seven experiments were pooled and analyzed as a single experiment with seven blocks.

Testing Diversity of Strains

To estimate the diversity of zwittermicin A and kanosamine B-producers, we sought to determine the minimum number of unique zwittermicin A and/or kanosamine B-producing strains in our collection. We considered isolates to be distinct strains only if phenotypic differences between them could be shown. Therefore, isolates were subjected to a series of phenotypic tests. All characterization was performed on isolates that had been colony purified on 1/2-strength TSA. To test for antibiotic resistance, isolates were streaked on 1/2-strength TSA containing tetracycline (5 μg/ml), neomycin (5 μg/ml), or chloramphenicol (1 μg/ml), and incubated at 28° C. overnight. Isolates that grew similarly when streaked in the presence or absence of antibiotic were classified as antibiotic resistant to test isolates for pigment production, they were grown on MES minimal medium at 28° C. for seven days and then scored visually. MES minimal medium contained 9.76 g/L 2- N-morpholine!ethan-sulfonic acid (MES), 2 g/L (NH₄)₂ SO₄, 0.2 g/L MgSO₄.7 H₂ O, 0.25 mg/L MnSO₄.7 H₂ O, 1.25 g/L K₂ HPO₄.3H₂ O, 2 g/L L-glutamic acid, 10 mg/L thiamine, 15 g/L agar, 40 mg/L FeCl₃.6H₂ O, 6 g/L sucrose and 1 mM of the amino acids threonine, serine, leucine, valine, and alanine, and was adjusted to pH 6.1. MES-Thr medium was MES minimal medium lacking threonine. We characterized the ability of isolates to grow on MES-Thr media by streaking isolates onto MES-Thr plates and incubating at 28° C. for four days and recording the rate of appearance of colonies for each strain. Phages ΦATCC 7064 and ΦATCC 27877 were obtained from the American Type Culture Collection and were propagated on bacterial strains ATCC 7064 and ATCC 27877, respectively. Phage Φ63 was propagated on strain Bt-1, and both Φ63 and Bt-1 were obtained from R. Landen. Sensitivity of isolates to phages Φ63, ¢ATCC7064 and ΦATCC27877 was determined by the soft-agar overlay method described above for P7, with plaque formation as the indicator of sensitivity.

Association of Zwittermicin A Production with P7^(s) and Eh⁺ Isolates

It was known that B. cereus strain UW85 produces two antibiotics, the novel aminopolyol, zwittermicin A, and kanosamine, that contribute to the suppression of alfalfa seedling damping-off. UW85 was originally identified in a labor-intensive screen for biological control activity. The study conducted above was intended to investigate whether sensitivity to P7 (P7^(s)) and the ability to inhibit E. herbicola (Eh⁺) were phenotypes that could be used to identify zwittermicin A producers and useful biocontrol strains.

4,307 B. cereus and B. thuringiensis isolates were screened for P7^(s) and/or Eh⁺ phenotypes. The isolates were obtained from geographically diverse soil samples collected at a total of 16 locations in five countries (Table 1 above), from alfalfa and soybean roots, and from stock culture collections (Table 2 above). The number of P7^(s) or P7^(r) Eh⁺ isolates identified from each source and the number of isolates tested was tabulated. P7^(s) isolates were identified in samples from 14 of the 16 soils examined as well as from alfalfa and soybean roots. Of the 87 P7^(s) isolates, all were Eh⁺ except SNY73 and LN100. P7^(r) Eh⁺ isolates were identified from each of the soils as well as from alfalfa roots. Among all the isolates tested, approximately 2% (85/4,307) of the isolates examined were P7^(s) Eh⁺ and 7% (132/1,876) were P7^(r) Eh⁺.

Alfalfa Field Stand Studies

Several of the most promising strains were then used as biocontrol inocula in alfalfa fields grown in Arlington, Hancock (Hah), Marshfield (Mar) and West Madison (WM), Wisconsin for the crop years 1993-1995. The results of those field trials are summarized in Tables 3.1, 3.2, 3.3, 4.1, 4.2 and 5 below.

From that data, it was determined that strain AS4-12 had the best overall field performance in most fields in enhancing the stand of alfalfa fields under normal cultivation conditions in the upper midwestern U.S. Also noteworthy, however was the performance of strain MS1-9. MS1-9 gave superior performance at the experimental fields at Marshfield, Wis. These particular fields have an endemic problem with severe rot root diseases of alfalfa, a problem which exhibited itself during this trial. MS1-9 was determined to be one of the best performers from among those screened under severe root rot disease conditions. The beneficial effects from MS1-9 manifested in both increased emergence of alfalfa seedlings as well as increased forage yield.

                  TABLE 3.1                                                        ______________________________________                                         Comparison of Bacillus cereus strains                                          1993-1994 Alfalfa Field Trials                                                 Emergence           1993                                                       (plants/m or %)     First harvest (g/m)                                        Treatment                                                                             Arl    Han     Mar  WM   Arl  Han   Mar  WM                             ______________________________________                                         Untreated                                                                             62     57.4    28   73   183  68    191  40                             Apron  56     77*     66*  87*  261  62    261  24                             US85   65     64      32   86*  232  80    236  52                             T30    --     --      --   --   --   --    --   --                             AP 17-5                                                                               72     44      29   68   237  51    211  31                             AP 2-1 59     43      30   71   206  53    221  33                             AP 4-12                                                                               89*    81*     65*  96*  154  71    288  45                             AS 23-15                                                                              58     47      31   81   241  62    218  21                             HP 20-2                                                                               53     40      31   82   209  52    219  36                             HP 24-2                                                                               75*    54      25   78   305  58    215  42                             HS 1-3 60     47      38*  71   240  60    223  38                             HS 18-4                                                                               66     62      34   75   221  76    212  41                             LP 34-16                                                                              65     55      37   85   311  70    246  23                             LP 38-2                                                                               64     52      35   83   349  47    229  22                             LP 4-13                                                                               63     57      30   76   285  74    218  30                             LP 15-3                                                                               60     52      31   74   228  67    203  28                             MS 1-9 61     53      38*  82   274  74    250  37                             MS 19-4                                                                               52     54      34   70   281  62    233  38                             WP 27-3                                                                               63     49      26   85   260  67    219  39                             WP 36-16                                                                              63     55      33   75   204  61    226  48                             WS 12-3                                                                               75*    51      23   64   246  62    186  25                             WS 12-4                                                                               68     51      35   75   256  47    227  41                             ______________________________________                                          *Significantly greater than untreated at P ≦0.10                         Significantly less than untreated at P ≦0.10                     

Alfalfa cultivar: Iroquois

Soil Types: Arlington; Plano silt loam Mancock; Plainfield loamy sand Marshfield; Withee silt loam West Madison; Plano silt loam

                  TABLE 3.2                                                        ______________________________________                                         Comparison of Bacillus cereus strains                                          1993-1994 Alfalfa Field Trials                                                 1993                1994                                                       Second harvest (g/m)                                                                               First harvest (g/m)                                        Treatment                                                                             Arl    Han     Mar  WM   Arl  Han   Mar  WM                             ______________________________________                                         Untreated                                                                             281    151     --   253  985  671   731  935                            Apron  346    137     --   256  962  596   826  868                            UW85   367    170     --   242  1117 762   919  924                            T30    --     --      --   --   --   --    --   --                             AP 17-5                                                                               325    153     --   215  934  642   786  888                            AP 2-1 301    160     --   223  920  624   872  940                            AS 4-12                                                                               303    154     --   248  1005 635   974  936                            AS 23-15                                                                              324    178     --   221  1058 735   847  865                            HP 20-2                                                                               302    129     --   240  1141 679   843  933                            HP 24-2                                                                               330    155     --   223  968  717   817  919                            HS 1-3 337    148     --   246  1008 667   825  931                            HS 18-4                                                                               296    161     --   230  1071 654   880  911                            HS 34-16                                                                              354    152     --   218  1022 653   884  885                            LP 38-2                                                                               434    119     --   185  1012 601   841  912                            LP 4-13                                                                               368    161     --   197  1091 684   775  915                            LPP 15-3                                                                              382    166     --   229  1201 654   810  929                            MS 1-9 379    153     --   220  1105 703   949  891                            MS 19-4                                                                               317    140     --   238  938  649   829  929                            WP 27-3                                                                               325    145     --   231  958  669   727  826                            WP 36-16                                                                              298    155     --   227  804  651   837  940                            WS 12-3                                                                               304    164     --   228  1074 644   780  941                            WS 12-4                                                                               373    128     --   258  1066 590   832  921                            ______________________________________                                    

There were no statistically significant differences due to treatment. (α=0.20)

                  TABLE 3.3                                                        ______________________________________                                         Comparison of Bacillus cereus strains                                          1993-1994 Alfalfa Field Trials                                                 1994                Final stand                                                Second harvest (g/m)                                                                               (plants/m or %)                                            Treatment                                                                             Arl    Han     Mar  WM   Arl  Han   Mar  WM                             ______________________________________                                         Untreated                                                                             465    479     526  522  28   39    28   45                             Apron  471    432     632* 440  26   53*   53*  45                             UW85   460    531     557  503  29   45    34*  44                             T30    --     --      --   --   --   --    --   --                             AP 17-5                                                                               466    478     580  495  --   --    --   --                             AP 2-1 459    368     598* 521  --   --    --   --                             AP 4-12                                                                               444    450     678* 517  34   56*   54*  51                             AS 23-15                                                                              454    482     452  499  --   --    --   --                             HP 20-2                                                                               480    438     540  530  --   --    --   --                             HP 24-2                                                                               462    551     550  440  --   --    --   --                             HS 1-3 481    431     556  500  --   --    --   --                             HS 18-4                                                                               428    453     583  466  --   --    --   --                             LP 34-16                                                                              448    427     577  499  --   --    --   --                             LP 38-2                                                                               505    423     525  517  --   --    --   --                             LP 4-13                                                                               447    405     533  463  --   --    --   --                             LP 15-3                                                                               480    453     551  465  --   --    --   --                             MS 1-9 442    475     602* 475  --   --    --   --                             MS 19-4                                                                               500    508     553  419  --   --    --   --                             WP 27-3                                                                               388    470     562  545  --   --    --   --                             WP 36-16                                                                              345    463     569  519  --   --    --   --                             WS 12-3                                                                               483    475     478  498  --   --    --   --                             WS 12-4                                                                               441    393     521  501  --   --    --   --                             ______________________________________                                          *Significantly greater than untreated P ≦0.2                             Significantly less than untreated                                       

                                      TABLE 4.1                                    __________________________________________________________________________     Comparison of Bacillus cereus strains                                          1994-1995 Alfalfa Field Trials                                                 Emergence     1994       1994                                                  (plants/m     First harvest                                                                             Second harvest                                        or %)         (g/m)      (g/m)                                                 Treatment                                                                           Arl                                                                              Han                                                                               Mar Arl                                                                               Han #                                                                               Mar                                                                               Arl                                                                               Han #                                                                               Mar                                           __________________________________________________________________________     Untreated                                                                           37                                                                               27 42  187                                                                               --   190                                                                               356                                                                               --   426                                           Apron                                                                               35                                                                               53*                                                                               79* 190                                                                               --   -- 291                                                                               --   --                                            UW85 37                                                                               23 51* 198                                                                               --   171                                                                               346                                                                               --   396                                           T30  38                                                                               29 46  176                                                                               --   187                                                                               287                                                                               --   395                                           AP 17-5                                                                             44                                                                               35*                                                                               47  207                                                                               --   183                                                                               314                                                                               --   374                                           AP 2-1                                                                              40                                                                               36*                                                                               50* 185                                                                               --   172                                                                               304                                                                               --   392                                           AP 4-12                                                                             29                                                                               33 48  148                                                                               --   192                                                                               283                                                                               --   413                                           AP 23-15                                                                            30                                                                               35*                                                                               50* 175                                                                               --   190                                                                               281                                                                               --   389                                           HP 20-2                                                                             32                                                                               35*                                                                               59* 165                                                                               --   194                                                                               250                                                                               --   415                                           HP 24-2                                                                             34                                                                               34*                                                                               49  188                                                                               --   205                                                                               316                                                                               --   425                                           HS 1-3                                                                              34                                                                               31 48  115                                                                               --   198                                                                               238                                                                               --   405                                           HS 18-4                                                                             34                                                                               26 52* 133                                                                               --   192                                                                               248                                                                               --   451                                           LP 34-16                                                                            38                                                                               32 51* 171                                                                               --   202                                                                               239                                                                               --   383                                           LP 38-2                                                                             35                                                                               34 51* 159                                                                               --   192                                                                               311                                                                               --   401                                           LP 4-13                                                                             31                                                                               38*                                                                               58* 181                                                                               --   212                                                                               331                                                                               --   441                                           LP 15-3                                                                             35                                                                               36*                                                                               47  173                                                                               --   183                                                                               297                                                                               --   404                                           MS 1-9                                                                              34                                                                               30 50* 182                                                                               --   200                                                                               335                                                                               --   429                                           MS 19-4                                                                             33                                                                               35*                                                                               51* 170                                                                               --   213                                                                               318                                                                               --   421                                           WP 27-3                                                                             29                                                                               28 55* 154                                                                               --   204                                                                               260                                                                               --   441                                           WP 36-16                                                                            33                                                                               28 40  141                                                                               --   173                                                                               249                                                                               --   425                                           WS 12-3                                                                             32                                                                               32 45  166                                                                               --   195                                                                               298                                                                               --   374                                           WS 12-4                                                                             33                                                                               36*                                                                               53* 144                                                                               --   188                                                                               299                                                                               --   365                                           __________________________________________________________________________      *Significantly greater than untreated control P ≦0.20                    Significantly less than untreated control                                     # The Hancock trial was replanted in the fall of 1994                    

Alfalfa cultivar: Iroquois

Soil Types: Arlington; Plano silt loam Hancock; Plainfield loamy sand Marshfield; Withee silt loam

                                      TABLE 4.2                                    __________________________________________________________________________     Comparison of Bacillus cereus strains                                          1994-1995 Alfalfa Field Trials                                                 1995          1995       Final Stand                                           First harvest Second harvest                                                                            (Plants/m                                             (g/m)         (g/m)      or %)                                                 Treatment                                                                           Arl                                                                               Han                                                                               Mar                                                                               Arl                                                                               Han #                                                                               Mar                                                                               Arl                                                                               Han #                                                                               Mar                                           __________________________________________________________________________     Untreated                                                                           688                                                                               529                                                                               1346                                                                              563                                                                               715  493                                                                               27.6                                                                              30.8 41.0                                          Apron                                                                               774                                                                               651                                                                               1109                                                                              571                                                                               861  432                                                                               27.8                                                                              49.6*                                                                               68.4*                                         UW85 774                                                                               573                                                                               1272                                                                              508                                                                               670  496                                                                               29.2                                                                              26.2 51.2*                                         T30  767                                                                               630                                                                               1122                                                                              551                                                                               840  446                                                                               -- --   --                                            AP 17-5                                                                             828                                                                               629                                                                               1247                                                                              559                                                                               770  456                                                                               -- --   --                                            AP 2-1                                                                              751                                                                               622                                                                               1256                                                                              426                                                                               809  506                                                                               -- --   --                                            AP 4-12                                                                             747                                                                               630                                                                               1195                                                                              517                                                                               806  437                                                                               22.4                                                                              38.4 53.0**                                        AP 23-15                                                                            832                                                                               644                                                                               1200                                                                              505                                                                               859  431                                                                               -- --   --                                            HP 20-2                                                                             720                                                                               574                                                                               1418                                                                              512                                                                               796  522                                                                               -- --   --                                            HP 24-2                                                                             793                                                                               638                                                                               1383                                                                              525                                                                               832  484                                                                               -- --   --                                            HS 1-3                                                                              777                                                                               571                                                                               1174                                                                              482                                                                               761  464                                                                               -- --   --                                            HS 18-4                                                                             725                                                                               538                                                                               1295                                                                              524                                                                               709  411                                                                               -- --   --                                            LP 34-16                                                                            822                                                                               680                                                                               1218                                                                              472                                                                               764  450                                                                               -- --   --                                            LP 38-2                                                                             951                                                                               617                                                                               1111                                                                              542                                                                               810  460                                                                               -- --   --                                            LP 4-13                                                                             667                                                                               676                                                                               1375                                                                              501                                                                               742  483                                                                               -- --   --                                            LP 15-3                                                                             741                                                                               645                                                                               1202                                                                              514                                                                               839  456                                                                               -- --   --                                            MS 1-9                                                                              729                                                                               548                                                                               1269                                                                              582                                                                               756  468                                                                               -- --   --                                            MS 19-4                                                                             842                                                                               566                                                                               1278                                                                              557                                                                               796  488                                                                               -- --   --                                            WP 27-3                                                                             688                                                                               604                                                                               1255                                                                              447                                                                               765  443                                                                               -- --   --                                            WP 36-16                                                                            822                                                                               554                                                                               1259                                                                              581                                                                               806  464                                                                               -- --   --                                            WS 12-3                                                                             856                                                                               604                                                                               1132                                                                              542                                                                               789  447                                                                               -- --   --                                            WS 12-4                                                                             769                                                                               656                                                                               1140                                                                              510                                                                               763  406                                                                               -- --   --                                            __________________________________________________________________________      # The Hancock trial was replanted in the fall of 1994.                   

                                      TABLE 5                                      __________________________________________________________________________     Comparison of Bacillus cereus strains                                          1995 Alfalfa Field Trials                                                      Establishment year                                                             Emergence     First harvest                                                                             Second harvest                                        (plants per   (fresh weight                                                                             (fresh weight                                         meter of row) in grams)  in grams)                                             Treatment                                                                           Arl                                                                              Han                                                                               Mar Arl                                                                               Han #                                                                               Mar                                                                               Arl                                                                               Han #                                                                               Mar                                           __________________________________________________________________________     Untreated                                                                           32                                                                               35 62  -- 403  301                                                                               512                                                                               636  1065                                          Apron                                                                               34                                                                               57*                                                                               72  -- 516  310                                                                               456                                                                               750  1037                                          Kodiak                                                                              36                                                                               33 75  -- 378  289                                                                               426                                                                               611  970                                           UW 85                                                                               22                                                                               37 86  -- 496  285                                                                               509                                                                               704  972                                           T 30 44                                                                               44 78  -- 523  283                                                                               533                                                                               789  945                                           AS 4-12                                                                             39                                                                               44 62  -- 459  314                                                                               524                                                                               779  1003                                          HP 20-2                                                                             18                                                                               38 67  -- 437  297                                                                               349                                                                               717  1025                                          HP 24-2                                                                             33                                                                               30 67  -- 363  293                                                                               449                                                                               655  928                                           LP 4-13                                                                             26                                                                               40 75  -- 415  319                                                                               482                                                                               696  989                                           MS 1-9                                                                              28                                                                               44 86  -- 560  291                                                                               390                                                                               770  1002                                          __________________________________________________________________________      *Significantly greater than the untreated control (P ≦0.2)               Significantly less than the untreated control                                 # The Hancock trial was replanted in the fall of 1994                          -- Not collected due to severe leafhopper damage                         

Alfalfa cultivar: Magnum III

Soil Types: Arlington; Plano silt loam Hancock; Plainfield loamy sand Marshfield; Withee silt loam 

We claim:
 1. A biologically pure culture of a bacteria having the identifying characteristics of Bacillus cereus MS1-9, ATCC
 55812. 2. An inoculum for application to alfalfa comprising a carrier and an effective quantity of a bacteria having the identifying characteristics of Bacillus cereus MS1-9, ATCC
 55812. 3. A method for protecting alfalfa in a growing medium from damping off disease comprising the steps of placing in the vicinity of the plant to be protected an effective quantity of a bacteria having the identifying charateristics of Bacillus cereus MS1-9, ATCC
 55812. 